The main types of fluorophores currently used as labels in diagnostic assays, such as immunoassays, are fluorecein, rhodamine, umbelliferone, and lanthanide chelates. Fluorescein is the most commonly used fluorophore since it is readily available in an activated form for direct coupling to antigens or antibodies. Both fluorescein and rhodamines show good chemical stability and have a proven record in actual use as labels. However, both fluorescein and the rhodamines have relatively small Stokes shifts, which limits the methodology that can be used in some respects. Both umbelliferones and lanthanide chelates have larger Stokes shifts, but their use as labels in immunoassays is relatively recent, and they are still untried in many respects. For example, the coupling between lanthanide chelates and antigens has presented some problems, and the fluorophore itself can be labile, which seriously restricts its use. Only a few assays have been published in which lanthanide chelates have been used as labels.
The problems seen with lanthanide chelates are indicative of the many constraints on the choice of fluorophore used in fluorescent labeling techniques. One constraint is the absorption and emission characteristic of the fluorophore, since many ligands, receptors, and materials associated with such compounds in the sample to be tested, e.g., blood, urine, or cerebrospinal fluid, will fluoresce and interfere with an accurate determination of the fluorescence of the fluorescent label. A second consideration is the quantum efficiency of the fluorophore, which should be high for good sensitivity. Another consideration is the ability to conjugate the fluorophore to ligands and receptors and the effect of such conjugation on the fluorophore. In many situations, conjugation to another molecule may result in a substantial change in the fluorescent characteristics of the fluorophore and, in some cases, substantially destroy or reduce the quantum efficiency of the fluorophore. Also of concern is whether the fluorescent molecules will interact with each other when in close proximity, resulting in self-quenching. An additional concern is whether there is non-specific binding of the fluorophore to other compounds or container walls, either by themselves or in conjunction with the compound to which the fluorophore is conjugated.
A recent development in the field of fluorescent labeling has been the use of phycobiliprotein conjugates Phycobiliproteins are a class of highly fluorescent proteins that form a part of the light-harvesting system in the photosynthetic apparatus of bluegreen bacteria and of two groups of eukaryotic algae, red algae and the cryptomonads. A particularly useful variation of their use comprises preparation of a phycobiliprotein tandem conjugate with a large Stokes shift. An example of such a conjugate is the covalent attachment of phycoerythrin to allophycocyanin. The resulting tandem conjugate has a large Stokes shift with an emission maximum at 660 nm and an excitation waveband that starts at about 440 nm. However, production of such tandem complexes requires the formation of a covalent or other chemical bond between the two components, therefore increasing the complexity of the production of the final conjugate.
Accordingly, there remains a need for fluorescent labels in diagnostic assays that have a large Stokes shift but which enjoy both stability and ease of preparation. Numerous techniques, such as histology, cytology, and immunoassays would enjoy substantial benefits from the availability of such a reagent.